Preservation of eukaryotic cells using reversible pore formation

Abstract

A method is disclosed for reliably stabilizing eukaryotic cells that express the P2X 7 receptor channel, particularly mammalian and other vertebrate cells, including human cells, for example mammalian macrophages, or hematopoietic stem cells, in order to introduce otherwise membrane-impermeable compounds that are helpful for stabilizing the cells during drying, chilling, freezing, freeze-drying, or cryopreservation. The cells are exposed to extracellular ATP in concentration sufficient to open pores in the plasma membrane. One or more otherwise membrane-impermeable compounds that aid the survivorship of cells are then introduced, for example, trehalose, and after a brief time the pores are closed—for example, by adding divalent cations, or by diluting the extracellular solution. Once the trehalose or other stabilizing compound has been introduced, the cells may be stably preserved. By taking advantage of an endogenous mammalian receptor and ATP, no antigenic compounds need be introduced.

1. A method for preserving vertebrate cells, said method comprising the steps of:
 (a) treating the vertebrate cells with a free extracellular agent comprising adenosine 5′-triphosphate or comprising 2′,3′-O-(benzoyl-4-benzoyl)-adenosine 5′-triphosphate, at a pH from about 6.0 to about 9.0; wherein the membranes of the cells comprise receptors that, in response to a sufficient concentration of the extracellular agent, cause the opening of pores in the membranes; and wherein the concentration of the extracellular agent is sufficient to cause the receptors to open pores in the membranes; whereby pores are opened in the membranes that would not be open in the absence of the extracellular agent; 
 (b) exposing the cells to a preservative; wherein the preservative crosses the cell membrane substantially faster while the pores are open than the preservative would cross the cell membrane under otherwise identical conditions without the opened pores; and wherein the preservative enhances viability if the cells are subsequently dehydrated, frozen, or freeze-dried; 
 (c) closing the pores by reducing the concentration of the free extracellular agent, wherein the pores are closed sufficiently soon after step (a) to maintain the viability of a substantial fraction of the cells; and 
 (d) dehydrating the cells, freezing the cells, or freeze-drying the cells; wherein the viability of the cells, if subsequently reconstituted or warmed, is substantially greater than would be the viability of otherwise identical cells that had not been subjected to said steps (a) through (d) prior to being dehydrated, frozen, or freeze-dried; 
 
       wherein:
 steps (a) and (b) may be conducted in either order, or steps (a) and (b) may be conducted simultaneously; provided that steps (a) and (b) overlap for a period of time sufficient for enough of the preservative to cross the cell membranes to enhance viability if the cells are subsequently dehydrated, frozen, or freeze-dried; and 
 steps (a) and (b) substantially precede step (c); and step (c) substantially precedes step (d); and 
 said vertebrate cells comprise nucleated cells or erythrocytes. 
 
     
     
       2. A method as recited in  claim 1 , wherein the cells are mammalian cells. 
     
     
       3. A method as recited in  claim 2 , wherein the cells are human cells. 
     
     
       4. A method as recited in  claim 2 , wherein the cells are erythrocytes. 
     
     
       5. A method as recited in  claim 1 , wherein the cells are nucleated cells. 
     
     
       6. A method as recited in  claim 1 , wherein in said pore-closing step the concentration of the free extracellular agent is reduced by diluting the extracellular agent, or by reacting the extracellular agent with a divalent cation, or by reacting the extracellular agent with a chelating agent, or by degrading the extracellular agent. 
     
     
       7. A method as recited in  claim 1 , wherein step (d) comprises dehydrating the cells. 
     
     
       8. A method as recited in  claim 1 , wherein step (d) comprises freezing the cells. 
     
     
       9. A method as recited in  claim 1 , wherein step (d) comprises freeze-drying the cells. 
     
     
       10. A method as recited in  claim 1 , wherein the receptors comprise P2X 7  receptors. 
     
     
       11. A method as recited in  claim 1 , wherein the cells are nucleated cells of a type whose membranes do not ordinarily comprise P2X 7  receptors; said method additionally comprising the step, prior to step (a), of introducing into the cells DNA encoding a P2X 7  receptor, and allowing the cells to express the P2X 7  receptor and to incorporate the P2X 7  receptor into the cell membranes; whereby, when step (a) is subsequently conducted, substantially more pores open in the cell membranes in response to the free extracellular agent than would have opened in otherwise identical cells with membranes lacking the P2X 7  receptor. 
     
     
       12. A method as recited in  claim 1 , wherein the preservative has a molecular weight less than about 900 Dalton. 
     
     
       13. A method as recited in  claim 1 , wherein the preservative comprises one or more compounds selected from the group consisting of trehalose, adenosine  5 ′-phosphate, adenosine 5′-phosphorothioate, sucrose, sorbitol, a disaccharide, an oligosaccharide, a metabolic activator, a metabolic inhibitor, sarcosine, octopine, taurine, proline, betaine, pinitol, ectoine, N-acetyl lysine, glycosylglycerate, and sulfotrehalose. 
     
     
       14. A method as recited in  claim 1 , wherein the preservative comprises trehalose. 
     
     
       15. A method as recited in  claim 1 , wherein the preservative comprises adenosine 5′-phosphorothioate. 
     
     
       16. A method as recited in  claim 1 , wherein step (a) is conducted at physiological temperature, and wherein step (b) is conducted at a substantially lower temperature that does not freeze the cells. 
     
     
       17. A method as recited in  claim 1 , wherein step (a) is conducted at about 37° C., and wherein step (b) is conducted at about 0° C. 
     
     
       18. A method as recited in  claim 1 , wherein the concentration of carbon dioxide during step (a) is not substantially above the ambient concentration of carbon dioxide. 
     
     
       19. A method as recited in  claim 1 , wherein step (a) is conducted at a pH from about 6.5 to about 8.0. 
     
     
       20. A method as recited in  claim 1 , wherein step (a) is conducted at a pH about 7.0. 
     
     
       21. A method as recited in  claim 1 , additionally comprising the step, after step (b), of adding an apoptosis inhibitor to the cells. 
     
     
       22. A method as recited in  claim 21 , wherein the apoptosis inhibitor comprises cyclosporin A or trifluoperazine. 
     
     
       23. A method as recited in  claim 1 , wherein the free extracellular agent comprises adenosine 5′-triphosphate. 
     
     
       24. A method as recited in  claim 1 , wherein the free extracellular agent comprises 2′,3′-O-(benzoyl-4-benzoyl)-adenosine 5′-triphosphate. 
     
     
       25. A method as recited in  claim 1 , additionally comprising a step, substantially after said pore-closing step, and substantially preceding step (d): of allowing the cells to recover under non-stressful conditions, for a time that is sufficiently long to enhance viability if the cells are subsequently dehydrated, frozen, or freeze-dried; and sufficiently short to retain enough preservative within the cells to enhance viability if the cells are subsequently dehydrated, frozen, or freeze-dried. 
     
     
       26. A method as recited in  claim 25 , wherein said recovering step occurs in a nutrient medium. 
     
     
       27. A method as recited in  claim 1 , additionally comprising the step, after step (d), of reconstituting or warming the preserved cells, and restoring the normal metabolism of at least some of the cells.